Conformation and ion-channeling activity of a 27-residue peptide modeled on the single-transmembrane segment of the IsK (minK) protein

IsK (minK) protein, in concert with another channel protein KVLQT1, mediates a distinct, slowly activating, voltage-gated potassium current across certain mammalian cell membranes. Site-directed mutational studies have led to the proposal that the single transmembrane segment of IsK participates in the pore of the potassium channel [Takumi, T. (1993) News Physiol. Sci. 8, 175-178]. We present functional and structural studies of a short peptide (K27) with primary structure NH2-1KLEALYILMVLGFFGFFTLGIMLSYI27R-COOH, corresponding to the transmembrane segment of IsK (residues 42-68). When K27 was incorporated, at low concentrations, into phosphatidylethanolamine, black-lipid membranes, single-channel activity was observed, with no strong ion selectivity. IR measurements reveal the peptide has a predominantly helical conformation in the membrane. The atomic resolution structure of the helix has been established by high-resolution 1H NMR spectroscopy studies. These studies were carried out in a solvent comprising 86% v/v 1,1,1,3,3,3-hexafluoro-isopropanol-14% v/v water, in which the IR spectrum of the peptide was found to be very similar to that observed in the bilayer. The NMR studies have established that residues 1-3 are disordered, while residues 4-27 have an alpha-helical conformation, the helix being looser near the termini and more stable in the central region of the molecule. The length (2. 6 nm) of the hydrophobic segment of the helix, residues 7-23, matches the span of the hydrocarbon chains (2.3 +/- 0.25 nm) of fully hydrated bilayers of phosphatidylcholine lipid mixture from egg yolk. The side chains on the helix surface are predominantly hydrophobic, consistent with a transmembrane location of the helix. The ion-channeling activity is believed to stem from long-lived aggregates of these helices. The aggregation is mediated by the pi-pi stacking of phenylalanine aromatic rings of adjacent helices and favorable interactions of the opposing aliphatic-like side chains, such as leucine and methionine, with the lipid chains of the bilayer. This mechanism is in keeping with site-directed mutational studies which suggest that the transmembrane segment of IsK is an integral part of the pore of the potassium channel and has a similar disposition to that in the peptide model system.     

Pharmacological activities of trimetoquinol and 1-benzyl halogen-substituted analogues on rat beta-adrenoceptor subtypes

The beta-adrenoceptor activity profile of trimetoquinol and its 1-benzyl halogen-substituted analogues was studied in rat tissues containing primarily beta 1 (atria)-, beta 2 (trachea)- and atypical beta/beta 3 (distal colon and brown adipose tissue)-adrenoceptors. Functional biological activity resided in the (-)-isomer of trimetoquinol which was 112-, 275-, 372- and 513-fold more potent than (+)-trimetoquinol in trachea, right atria, distal colon and brown adipose tissue, respectively. (+/-)-Trimetoquinol was equally or slightly less active than (-)-trimetoquinol. The 1-benzyl halogen-substituted analogues of trimetoquinol exhibited differential activation of beta-adrenoceptor subtypes. In functional assays, 3'-iodotrimetoquinol was a potent activator of all beta-adrenoceptor subtypes. 3',5'-Diiodotrimetoquinol was 10-fold more potent as an agonist in tissues containing atypical beta/beta 3-adrenoceptors than those tissues containing beta 1- and beta 2-adrenoceptor sites. Furthermore, this drug was a partial agonist as compared to (+/-)-trimetoquinol and 3'-iodotrimetoquinol on beta 1-adrenoceptors. Pharmacological properties of the compounds on rat beta 3-adrenoceptors expressed in Chinese hamster ovary (CHO) cells were consistent with results observed in functional assays. 3',5'-Diiodotrimetoquinol possessed the greatest potency for activation of adenylyl cyclase. Rank order of affinity for rat beta 3-adrenoceptor was 3'-iodotrimetoquinol = 3',5'-diiodotrimetoquinol > (+/-)-trimetoquinol > (-)-isoprenaline. These results suggest that 3',5'-diiodotrimetoquinol is a promising drug for further chemical modification in the development of selective beta 3-adrenoceptor ligands.     

Multiple isotope effects as a probe of proton and hydride transfer in the 6-phosphogluconate dehydrogenase reaction

Primary solvent deuterium, primary substrate deuterium, multiple solvent deuterium/substrate deuterium, and multiple solvent deuterium/13C isotope effects on V/K6PG have been measured for the Candida utilis and sheep liver 6-phosphogluconate dehydrogenases (6PGDH). Proton inventory data suggest the presence of a significant medium effect in a step preceding hydride transfer and the presence of a kinetic solvent deuterium isotope effect on hydride transfer. Multiple isotope effect data confirm the presence of multiple solvent deuterium sensitive steps, likely including a conformational change preceding hydride transfer, hydride transfer, and decarboxylation.     

Cloning and recombinant expression of a barred sand bass (Paralabrax nebulifer) cDNA. The encoded protein displays structural homology and immunological crossreactivity to human complement/cofactor related plasma proteins

A new cofactor related cDNA in the bony fish Paralablax nebulifer, (barred sand bass) was isolated from a sand bass liver cDNA library. The clone (c71) is 1040 bp in size and the predicted translation product of 204 amino acids contains a hydrophobic signal peptide, which is followed by a region of three short consensus repeats (SCRs). The three SCRs display high homology to SCRs of the 110 kDa chain of the sand bass plasma cofactor protein, and to a lesser degree to human complement factor H related protein 3 (FHR-3) and to human factor H. Recombinant expression of the c71 cDNA in the baculovirus system shows a product of an apparent molecular mass of 27 kDa, which is secreted and glycosylated. It also contains a His-tag for purification purposes. Removal of the His-tag yields a 24 kDa protein, and deglycosylation further reduces the molecular mass to 21 kDa. This size is in agreement with the calculated molecular mass based on amino acid composition. The sand bass SBCFR-1 protein is immunologically related to the human complement proteins, factor H and factor H-related protein 3. The recombinantly expressed protein reacted with antisera against the human FHR-3 protein and SCRs 19-20 of human factor H. The presence of SCR-containing proteins in sand bass plasma and their structural and immunological homology to human FHR-3 and factor H suggests for a common function between these evolutionary related proteins.     

Mutations in the SDHB gene are associated with extra-adrenal and/or malignant phaeochromocytomas

Germ-line mutations in the genes encoding succinate dehydrogenase complex subunits B (SDHB) and D (SDHD) have been reported in familial paragangliomas and apparently sporadic phaeochromocytomas (ASP), but the genotype-phenotype relationships of these mutations are unknown. Eighty-four patients (all but 2 followed up for 8.8 +/- 5.7 years) with ASP (57 with adrenal tumors, 27 with extra-adrenal, multiple, malignant, or recurrent tumors) were screened for the major susceptibility genes for phaeochromocytoma (RET, VHL, SDHD, and SDHB). Thirty-three tumors were available for molecular analysis, enzyme assays, and immunohistochemistry. No (0%) RET and 2 (2.4%) VHL mutations were detected. Only two coding single nucleotide polymorphisms in the SDHD gene (G12S and H50R) were found in 6 patients (7%). Conversely, six deleterious mutations in the SDHB gene were identified in 8 patients (9.5%). Ectopic site and recurrence or malignancy were strongly associated with SDHB mutations (7 of 8, 87%, versus 20 of 76, 26%; P = 0.001). Somatic DNA analysis indicated a loss of heterozygosity at chromosome 1p36 (SDHB locus) in 16 of 33 cases (48%). A loss of heterozygosity at the SDHB locus was found in all tumors with SDHB mutation, and assays of respiratory chain enzymes showed a complete loss of complex II catalytic activity. The vascular architecture of tumors with SDHB mutations displayed features typical of malignancy. These data strongly suggest that SDHB gene is a tumor suppressor gene and that the identification of germ-line mutations in SDHB gene in patients with ASPs should be considered as a high-risk factor for malignancy or recurrence.     

Circadian rhythms in human body composition

The study investigates how the human body composition (BC) changes as a function of the day-night cycle. The BC was investigated using bioelectrical impedance analysis (BIA) of 10 clinically healthy subjects (CHS), monitored in supine position (readings at 2-h intervals), avoiding mealtimes, dietary abuses, and bladder and intestinal retention. Time series data were analyzed for their temporal characteristics and circadian rhythm (CR). All the variables of BC (lean body mass, fat body mass, body cell mass, total body water, intracellular and extracellular body water, sodium and potassium exchangeable pool) showed a within-day variability with nighttime crests. Such an oscillatory synchronism corroborates the hypothesis that the rest time plays a fundamental role, via its anabolic effects, in conferring the nocturnal phase to the CR of the human BC.     

Heterologous expression of an engineered truncated form of human Lewis fucosyltransferase (Fuc-TIII) by the methylotrophic yeast Pichia pastoris

A stable GS115 Pichia pastoris recombinant strain was constructed to secrete a truncated form of the human alpha(1,3/4) fucosyltransferase (amino acids 45-361). Enzyme production resulted from a secretory pathway based on the pre-pro- alpha mating factor signal sequence of the yeast Saccharomyces cerevisiae . Following its transit through the Golgi apparatus, the enzyme accumulated in the periplasmic space before its release in the culture broth (about 30 mg/l). Cell-enclosed enzyme ( approximately 0.16%) proved to be fairly stable for many freezing and thawing cycles and could be used several times as an immobilized catalyst. Soluble enzyme (>99.8%) representing the main protein of the culture broth (10%) has been characterized by Western-blotting, substrate specificities and kinetic parameters. The two forms (cell-enclosed and soluble) of recombinant enzyme may be used for in vitro synthesis of Lewisadeterminants.